Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

Agave REVEILLE1 regulates the onset and release of seasonal dormancy in Populus.

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.

Insights into the complicated networks contribute to adventitious rooting in transgenic MdWOX11 apple microshoots under nitrate treatments.

Adventitious root formation is a bottleneck for the mass propagation of microshoots, and nitrate is an essential nutrient regulating adventitious roots. WOX11 is involved in adventitious rooting. But the crosstalk between nitrate and WOX11 is completely unknown. In this study, MdWOX11 transgenic apple microshoots were grown on different nitrate treatments. Low nitrate promotes adventitious rooting in overexpressed microshoots more than wild type and RNA interference microshoots. In contrast, medium nitrate significantly inhibits it in overexpressed and RNA interference microshoots compared with wild type microshoots. Stem anatomy indicated that medium nitrate delays root primordia formation compared with low nitrate. Methyl jasmonate and zeatin riboside played positive and negative roles in adventitious rooting, respectively. Transcriptomic analysis was conducted to understand the molecular mechanisms behind the phenotypes better. Hormone signalling, sugar metabolism, nitrogen metabolism, cell cycle and root development pathway-related genes were selected for their potential involvement in adventitious rooting. Results suggest that nitrogen signaling and MdWOX11 expression affect cytokinin accumulation and response to cytokinin through regulating the expression of genes related to cytokinin synthesis and transduction pathways, which ultimately affect adventitious rooting. This study provided important insights into the complicated networks involved in adventitious rooting in transgenic microshoots under nitrate treatments.

Transcriptome analysis reveals the regulatory mechanism by which MdWOX11 suppresses adventitious shoot formation in apple.

Adventitious shoot (AS) regeneration accelerates plant reproduction and genetic transformation. WOX11 is involved in many biological processes, but its regulation of AS regeneration has not been reported. Here, we showed that the genotype and CK/IAA ratio of apple leaves were the key factors that affected their capacity for AS formation. Moreover, the expression level of MdWOX11 was negatively correlated with the capacity for AS formation. Phenotypic analysis of MdWOX11 transgenic plants showed that overexpression of MdWOX11 inhibited AS formation. Endogenous hormone analysis demonstrated that the contents of auxin (IAA), cytokinin (CK), and abscisic acid (ABA) were higher in MdWOX11-RNAi plants than in MdWOX11-OE transgenic plants. We used RNA sequencing to examine the transcriptional responses of genes in MdWOX11-RNAi and MdWOX11-OE transgenic apple plants at different AS stages. We identified 8066 differentially expressed genes and focused our analysis on those involved in the IAA, CK, ABA, and gibberellin (GA) hormone signaling pathways. The expression of genes related to the CK signaling pathway and shoot development was higher in GL-3 than in MdWOX11-OE transgenic plants during the callus and AS emergence stages. However, the expression of MdCKX5 was higher in MdWOX11-OE transgenic plants than in GL3 and MdWOX11-RNAi transgenic plants. Yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and ChIP-qPCR showed that MdWOX11 binds to the promoter of MdCKX5, and a dual-luciferase reporter assay showed that MdWOX11 enhanced the promoter activity of MdCKX5. We concluded that MdCKX5 acts downstream of MdWOX11 to control AS formation, and we built a regulatory model of the suppression of AS formation by MdWOX11 in apple.

Transcriptome analysis reveals that cytokinins inhibit adventitious root formation through the MdRR12-MdCRF8 module in apple rootstock.

Adventitious root (AR) formation is great significance for apple rootstock breeding. Transcriptome analyses were performed with cytokinins (CTKs) signal treatments to analyze the mechanism of AR formation. The results showed that 6-benzyadenine (6-BA) treatment inhibited AR formation. Histological analysis also observed that AR primordium cell formation was significantly suppressed by 6-BA treatment; the ratio of auxin/cytokinins exhibited the lowest values at 1 and 3 day (d) in the 6-BA treatment group. Furthermore, the differentially expressed genes were divided into five categories, including auxin, cytokinins, other hormones, cell cycle, and carbohydrate metabolism pathways. Due to the study of cytokinins signal treatment, it is important to understand the particular module mediated by the cytokinins pathway. The expression level of MdRR12 (a family member of B-type cytokinins-responsive factors) was significantly upregulated at 3 d by 6-BA treatment. Compared to the wild type, the 35S::MdRR12 transgenic tobaccos suppressed AR formation. The promoter sequence of MdCRF8 contains AGATT motif elements that respond to MdRR12. RNA-seq and RT-qPCR assays predicted cytokinins response factor (MdCRF8) to be a downstream gene regulated by MdRR12. The activity of the pro-MdCRF8-GUS promoter was obviously induced by 6-BA treatment and inhibited by lovastatin (Lov) treatment. Yeast one-hybrid, dual-luciferase reporter, and GUS coexpression assays revealed that MdRR12 could directly bind to the MdCRF8 promoter. Additionally, 35S::MdCRF8 transgenic tobaccos also blocked AR growth. Compared to the wild type, 35S::MdRR12 and 35S::MdCRF8 transgenic tobaccos enhanced sensitivity to cytokinins. Thus, we describe that MdRR12 and MdCRF8 function as integrators of cytokinins signals that affect cell cycle- and carbohydrate metabolism-related genes to regulate cell fate transition during AR formation. On the basis of these results, we concluded that the MdRR12-MdCRF8 module is involved in the negative regulation of AR formation in apple rootstock and can potentially be applied in agriculture using genetic approaches.

Transcriptome Analysis Reveals Multiple Genes and Complex Hormonal-Mediated Interactions with PEG during Adventitious Root Formation in Apple.

Adventitious root (AR) formation is a bottleneck for the mass propagation of apple rootstocks, and water stress severely restricts it. Different hormones and sugar signaling pathways in apple clones determine AR formation under water stress, but these are not entirely understood. To identify them, GL-3 stem cuttings were cultured on polyethylene glycol (PEG) treatment. The AR formation was dramatically decreased compared with the PEG-free control (CK) cuttings by increasing the endogenous contents of abscisic acid (ABA), zeatin riboside (ZR), and methyl jasmonate (JA-me) and reducing the indole-3-acetic acid (IAA) and gibberellic acid 3 (GA3) contents. We performed a transcriptomic analysis to identify the responses behind the phenotype. A total of 3204 differentially expressed genes (DEGs) were identified between CK and PEG, with 1702 upregulated and 1502 downregulated genes. Investigation revealed that approximately 312 DEGs were strongly enriched in hormone signaling, sugar metabolism, root development, and cell cycle-related pathways. Thus, they were selected for their possible involvement in adventitious rooting. However, the higher accumulation of ABA, ZR, and JA-me contents and the upregulation of their related genes, as well as the downregulation of sugar metabolism-related genes, lead to the inhibition of ARs. These results indicate that AR formation is a complicated biological process chiefly influenced by multiple hormonal signaling pathways and sugar metabolism. This is the first study to demonstrate how PEG inhibits AR formation in apple plants.

Transcriptome analysis reveals the inhibitory nature of high nitrate during adventitious roots formation in the apple rootstock.

In the process of vegetative propagation of apple rootstocks, the development of adventitious roots (ARs) has crucial importance. Nitrate is an essential nutrient necessary for plant growth; however, the inhibitory effect of high nitrate on ARs formation has not been explored. The physiological and molecular mechanisms underlying ARs inhibition were examined in this study. Stem cuttings of B9 apple rootstock were cultured on two nitrate treatments (T1 = 18.7 mM L-1 and T2 = 37.5 mM L-1 ), where T2 was identified as ARs inhibiting treatment. Morphological and anatomical observations advocating that high availability of nitrate inhibited AR formation by delaying the ARs initiation and emergence stages, where the root number was 287%, and the length was 604.6% lower than the T1 cuttings. Moreover, the contents of endogenous hormones were also elevated in response to T2 at most of the time points, which may cause a hormonal imbalance within the plant body and drive toward ARs inhibition. Furthermore, 3686 genes were differentially expressed by high-throughput sequencing. Out of these, 1797 genes were upregulated, and 1889 genes were downregulated. Approximately 238 genes related to nitrate, hormones, root development, and cell-cycle induction pathways were selected according to their potential to be involved in ARs regulation. This is the first study providing information regarding the inhibitory effect of high nitrate on ARs formation in apple rootstock.

Mdm-MIR393b-mediated adventitious root formation by targeted regulation of MdTIR1A expression and weakened sensitivity to auxin in apple rootstock.

Adventitious root (AR) formation is of great significance for apple rootstock breeding. It is widely accepted that miR393 influences AR formation in many plant species; however, the molecular mechanism by which factors regulate AR formation remains insufficient. In this study, the evolutionary relationship of mdm-miR393 and candidate target genes MdTIR1/AFB was systematically identified, and the expression patterns were analysed. Multisequence alignment analysis of miR393 family members suggests that miR393 conservatively evolved between different species. The evolutionary relationship of the TIR1/AFBs can be divided into G1, G2 and G3 subgroups. During AR formation, the expression level of mdm-miR393a/b/c was significantly upregulated at 1 d and 7 d by exogenous auxin treatment. Furthermore, the expression levels of MdTIR1A, MdTIR1D, MdAFB1, MdAFB2, MdAFB3, MdAFB4 and MdAFB8 also appeared to be significantly changed by exogenous auxin induction. Subsequently, tissue-specific expression analysis showed that the expression levels of mdm-miR393 and MdTIR1/AFBs in different tissues exhibited significant differences. The promoter of mdm-miR393 contains multiple elements that respond to ABA, adversity and light signals; auxin treatment can activate the mdm-MIR393b promoter but is obviously inhibited by NPA treatment. The targeting relationship between mdm-MIR393b and MdTIR1A was verified by expression patterns, degradation group data, transient tobacco conversion results, and genes functions experiments. Heterologous overexpression of mdm-MIR393b (35S::mdm-MIR393b) decreased the number of ARs in the phenotype and reduced the expression level of the target gene NtTIR1 in tobacco. Compared to the wild type, the 35S::mdm-MIR393b transgenic plants demonstrated insensitivity to auxin. Furthermore, tir1 mutant exhibited reduced root system structure relative to the control. The above results illustrated that mdm-MIR393b is involved in mediating AR formation by targeted regulation of MdTIR1A expression in apple rootstock.

Melatonin promotes adventitious root formation in apple by promoting the function of MdWOX11.

BACKGROUND: Melatonin (MT) is important for plant growth and development; however, it is not known whether MT is involved in apple adventitious root (AR) development. In this study, we treated Malus prunifolia (MP) at four different stages of AR development, and analyzed the level of the endogenous hormones MT, auxin (IAA), zeatin-riboside (ZR), abscisic acid (ABA), and gibberellins (GA1 + 3) in all four treatment groups and the untreated control group. The expression of MT, IAA biosynthesis, transport and signal transduction, the cell cycle, and root development related genes were quantified by RT-qPCR. The function of MdWOX11 was analyzed in transgenic apple plants. RESULTS: The promotion of AR development by MT was dependent on the stage of AR induction between 0 and 2 d in apple rootstocks. MT-treatment increased the level of IAA and crosstalk existed between MT and IAA during AR formation. The expression of MdWOX11 was induced by MT treatment and positively regulated AR formation in apple. Furthermore, transgenic lines that overexpressed MdWOX11 lines produced more ARs than 'GL3'. Phenotypic analysis indicated that MdWOX11 overexpression lines were more sensitive to exogenous MT treatment than 'GL3', suggesting that MdWOX11 regulates AR formation in response to MT in apple rootstock. CONCLUSIONS: MT promotes AR formation mainly during the AR induction stage by inducing IAA levels and upregulating MdWOX11.

Genome-wide identification and expression profiling of the YUCCA gene family in Malus domestica.

The plant hormone auxin is essential for plant growth and development. YUCCA proteins catalyse the rate-limiting step for endogenous auxin biosynthesis. In this study, we isolated 20 MdYUCCA genes from apple genome. MdYUCCA6a, MdYUCCA8a, and MdYUCCA10a were expressed in most organs and could support whole plant basal auxin synthesis. MdYUCCA4a, MdYUCCA10b, and MdYUCCA11a expression indicated roles for these genes in auxin biosynthesis in vegetative organs. MdYUCCA2b, MdYUCCA11b, and MdYUCCA11d were mainly expressed in flower organs. High temperature induced the expression of MdYUCCA4a, MdYUCCA6a, MdYUCCA8a, and MdYUCCA10a, and down-regulated the expression of MdYUCCA2b and MdYUCCA6b. Dual-luciferase assay indicated that MdPIF4 could trans-activate the MdYUCCA8a promoter. Overexpression of MdYUCCA8a increased IAA content, increased stem height, enhanced apical dominance, and led to silique malformation. These results provide a foundation for further investigation of the biological functions of apple MdYUCCAs.

miRNAs associated with auxin signaling, stress response, and cellular activities mediate adventitious root formation in apple rootstocks.

Adventitious root (AR) formation is essential for the vegetative propagation of apple rootstocks. miRNAs play a significant role in regulating AR development, however, large-scale transcriptomic data on miRNA mediated AR formation in apple rootstocks is lacking. Therefore, in order to identify the molecular mechanisms underlying AR formation in 'M9-T337' apple rootstocks, transcriptomic changes occurring during key time points of AR formation (0, 3, and 16 days) were analyzed using high-throughput sequencing with a focus on miRNAs. A total of 84 known miRNAs and 56 novel miRNAs have differentially expressed were identified. Additionally, a total of 88 target genes of known miRNAs and 76 target genes of novel miRNAs were identified by degradome sequencing. The expression levels of the miRNAs and target genes were quantified by RT-qPCR. Results indicate that miRNAs and their target genes are associated with auxin signal-related (miR160 and miR390), stress response-related (miR398, miR395 and miR408), cell fate transformation-, proliferation- and enlargement-related (miR171, miR156, miR166, miR319 and miR396). These all involve pathways that participate in AR formation in 'M9-T337' apple rootstock. In addition, hormones (AUX, CTK, GA3, BR, JA, and ABA) are also involved in regulating AR formation. The candidate genes belonging to pathways associated with AR formation exhibited significantly higher expression levels, providing evidence that they may be involved in the regulation of AR development. The collective results of the present study indicate that the developmental process associated with AR formation in apple rootstock is extremely complex. The known and novel miRNAs and target genes that were identified by high-throughput and degradome sequencing, respectively, provide a framework for the future analysis of miRNAs associated with AR development in apple rootstocks, and provide new information that can be used to better understand AR development in woody plants.

Inhibition of adventitious root development in apple rootstocks by cytokinin is based on its suppression of adventitious root primordia formation.

Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l-1 of exogenous 6-benzyl adenine (6-BA) was administered to 3 and 7-day-old apple rootstocks 'M.26' cuttings (3 and 7 days 6-BA) by transferring them from a rooting medium containing indole-3-butanoic acid to the medium containing 6-BA. Anatomical and morphological observations revealed that the exogenous application of 6-BA inhibited primordia formation in the 3 days 6-BA but not the 7 days 6-BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6-BA than in 7 days 6-BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6-BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6-BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3-7 days is the time period in which AR primordia formation occurs in cuttings of 'M.26' and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3-7 days period after culturing in rooting medium.

Genome-wide identification of SERK genes in apple and analyses of their role in stress responses and growth.

BACKGROUND: Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases associated with various signaling pathways. These kinases have a relationship with stress signals, and they are also believed to be important for regulating plant growth. However, information about this protein family in apple is limited. RESULTS: Twelve apple SERK genes distributed across eight chromosomes were identified. These genes clustered into three distinct groups in a phylogenetic analysis. All of the encoded proteins contained typical SERK domains. The chromosomal locations, gene/protein structures, synteny, promoter sequences, protein-protein interactions, and physicochemical characteristics of MdSERK genes were analyzed. Bioinformatics analyses demonstrated that gene duplications have likely contributed to the expansion and evolution of SERK genes in the apple genome. Six homologs of SERK genes were identified between apple and Arabidopsis. Quantitative real-time PCR analyses revealed that the MdSERK genes showed different expression patterns in various tissues. Eight MdSERK genes were responsive to stress signals, such as methyl jasmonate, salicylic acid, abscisic acid, and salt (NaCl). The application of exogenous brassinosteroid and auxin increased the growth and endogenous hormone contents of Malus hupehensis seedlings. The expression levels of seven MdSERK genes were significantly upregulated by brassinosteroid and auxin. In addition, several MdSERK genes showed higher expression levels in standard trees of 'Nagafu 2' (CF)/CF than in dwarf trees of CF/'Malling 9' (M.9), and in CF than in the spur-type bud mutation "Yanfu 6" (YF). CONCLUSION: This study represents the first comprehensive investigation of the apple SERK gene family. These data indicate that apple SERKs may function in adaptation to adverse environmental conditions and may also play roles in controlling apple tree growth.

Genome-wide identification and expression analysis of brassinosteroid biosynthesis and metabolism genes regulating apple tree shoot and lateral root growth.

In plants, brassinosteroid biosynthesis and metabolism genes affect endogenous brassinosteroid metabolic processes as well as stem and root growth. However, there is little information available regarding these genes in apple. In this study, 22 brassinosteroid biosynthesis and metabolism genes were identified in apple (Malus domestica). These genes were named according to their chromosomal locations and the Arabidopsis thaliana homologs. Their conserved characteristic domains, evolutionary relationships, syntenic relationships, chemical characteristics, gene/protein structures, interactions among the encoded proteins, promoter sequences, and functions were investigated. These 22 genes were clustered with their A. thaliana homologs based on bioinformatics analyses, which suggested they are functionally similar in apple and A. thaliana. Tissue-specific expression levels revealed that most of these genes are important for stem growth and development, while several of these genes affect lateral root formation. The transcription patterns of these genes in shoot tips were investigated following diverse treatments [brassinosteroid (shoot tips and roots), brassinazole, auxin, and temperature]. Gene expression levels were also examined in different grafting combinations ('Nagafu No. 2'/Malling 9 and 'Nagafu No. 2'/'Nagafu No. 2') and shoot varieties ('Yanfu No. 6' and 'Nagafu No. 2'). The results indicated that these genes may be involved in apple stem and root growth. The comprehensive genome-wide analysis of brassinosteroid biosynthesis and metabolism genes presented herein may be useful for breeding new apple cultivars with increased vigor. The data also represent a rich genetic resource for future apple gene functional investigations that may have implications for the genetic improvement of apple tree species.

Transcriptome Analysis Reveals Multiple Hormones, Wounding and Sugar Signaling Pathways Mediate Adventitious Root Formation in Apple Rootstock.

Adventitious roots (AR) play an important role in the vegetative propagation of apple rootstocks. The potential role of hormone, wounding, and sugar signalling pathways in mediating AR formation has not been adequately explored and the whole co-expression network in AR formation has not been well established in apple. In order to identify the molecular mechanisms underlying AR formation in 'T337' apple rootstocks, transcriptomic changes that occur during four stages of AR formation (0, 3, 9 and 16 days) were analyzed using high-throughput sequencing. A total of 4294 differentially expressed genes were identified. Approximately 446 genes related to hormones, wounding, sugar signaling, root development, and cell cycle induction pathways were subsequently selected based on their potential to be involved in AR formation. RT-qPCR validation of 47 genes with known functions exhibited a strong positive correlation with the RNA-seq data. Interestingly, most of the candidate genes involved in AR formation that were identified by transcriptomic sequencing showed auxin-responsive expression patterns in an exogenous Indole-3-butyric acid (IBA)-treatment assay: Indicating that endogenous and exogenous auxin plays key roles in regulating AR formation via similar signalling pathways to some extent. In general, AR formation in apple rootstocks is a complex biological process which is mainly influenced by the auxin signaling pathway. In addition, multiple hormones-, wounding- and sugar-signaling pathways interact with the auxin signaling pathway and mediate AR formation in apple rootstocks.

iTRAQ-Based Proteomic Analysis Reveals Potential Regulation Networks of IBA-Induced Adventitious Root Formation in Apple.

Adventitious root (AR) formation, which is controlled by endogenous and environmental factors, is indispensable for vegetative asexual propagation. However, comprehensive proteomic data on AR formation are still lacking. The aim of this work was to study indole-3-butyric acid (IBA)-induced AR formation in the dwarf apple rootstock 'T337'. In this study, the effect of IBA on AR formation was analysed. Subsequent to treatment with IBA, both the rooting rate and root length of 'T337' increased significantly. An assessment of hormone levels in basal stem cuttings suggested that auxin, abscisic acid, and brassinolide were higher in basal stem cuttings that received the exogenous IBA application; while zeatin riboside, gibberellins, and jasmonic acid were lower than non-treated basal stem cuttings. To explore the underlying molecular mechanism, an isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic technique was employed to identify the expression profiles of proteins at a key period of adventitious root induction (three days after IBA treatment). In total, 3355 differentially expressed proteins (DEPs) were identified. Many DEPs were closely related to carbohydrate metabolism and energy production, protein homeostasis, reactive oxygen and nitric oxide signaling, and cell wall remodeling biological processes; as well as the phytohormone signaling, which was the most critical process in response to IBA treatment. Further, RT-qPCR analysis was used to evaluate the expression level of nine genes that are involved in phytohormone signaling and their transcriptional levels were mostly in accordance with the protein patterns. Finally, a putative work model was proposed. Our study establishes a foundation for further research and sheds light on IBA-mediated AR formation in apple as well as other fruit rootstock cuttings.

Transcriptome analysis reveals the effects of sugar metabolism and auxin and cytokinin signaling pathways on root growth and development of grafted apple.

BACKGROUND: The root architecture of grafted apple (Malus spp.) is affected by various characteristics of the scions. To provide information on the molecular mechanisms underlying this influence, we examined root transcriptomes of M. robusta rootstock grafted with scions of wild-type (WT) apple (M. spectabilis) and a more-branching (MB) mutant at the branching stage. RESULTS: The growth rate of rootstock grafted MB was repressed significantly, especially the primary root length and diameter, and root weight. Biological function categories of differentially expressed genes were significantly enriched in processes associated with hormone signal transduction and intracellular activity, with processes related to the cell cycle especially down-regulated. Roots of rootstock grafted with MB scions displayed elevated auxin and cytokinin contents and reduced expression of MrPIN1, MrARF, MrAHP, most MrCRE1 genes, and cell growth-related genes MrGH3, MrSAUR and MrTCH4. Although auxin accumulation and transcription of MrPIN3, MrALF1 and MrALF4 tended to induce lateral root formation in MB-grafted rootstock, the number of lateral roots was not significantly changed. Sucrose, fructose and glucose contents were not decreased in MB-grafted roots compared with those bearing WT scions, but glycolysis and tricarboxylic acid cycle metabolic activities were repressed. Root resistance and nitrogen metabolism were reduced in MB-grafted roots as well. CONCLUSIONS: Our findings suggest that root growth and development of rootstock are mainly influenced by sugar metabolism and auxin and cytokinin signaling pathways. This study provides a basis that the characteristics of scions are related to root growth and development, resistance and activity of rootstocks.